Exercise 6 Frog Embryology - 1

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Exercise 6 Study of Prepared Sides of the Developmental Stages of Frog

EXERCISE 6
STUDY OF PREPARED SLIDES OF
THE DEVELOPMENTAL STAGES
OF FROG

Structure
6.1 Introduction Features of Tadpole of a Frog:
Whole Mount
Objectives
Features of Transverse Section
6.2 Material Required
of Tadpole of through the Eye
6.3 Study Guide Region

6.4 Development Stages of Frog Features of Transverse Section


of Tadpole through the Auditory
Unfertilized Egg of Frog
Vesicles (Ears)
Zygote: Fertilized Egg
Features of Transverse Section
Blastula of Tadpole through the Head
and Gills
Gastrula

Neurula
6.5 Terminal Questions

6.1 INTRODUCTION
Animal development in vertebrates begins usually with the fertilization of a
haploid egg by a haploid sperm. The nuclei of the egg and sperm fuse, and
form a zygote which similar to the parents is diploid. The genes contributed by
the male and female parents to their respective male and female gametes
determine the characteristics of the zygote offspring. Compared to other
biological processes, embryonic development is relatively slow as new cells,
tissues and organs make their appearance in the embryo over a period of
hours, days or weeks. In this laboratory exercise, we shall study the
developmental stages of frog , starting from the unfertilized egg upto the
tadpole stage, with the help of permanent slides of whole mounts and stained
sections. We will begin this study from the unfertilized egg upto the tadpole
stage. 65
BZYCL-134 Laboratory of Comparative Anatomy and Development Biology of Vertebrates

Objectives
After completing this laboratory exercise you should be able to:
 identify and describe the representative stages in frog development
from fertilized egg upto tadpole stage from permanent, prepared slides
studied under the compound microscope, and
 draw and label whole mounts and sections of developmental stages of
frogs from the prepared slides provided.

6.2 MATERIAL REQUIRED


Prepared permanent microscope slides of the following developmental
stages of frog:

• whole mount of unfertilized egg


• whole mount of blastula and its vertical section (VS)
• whole mount of gastrula and its vertical section (VS)
• whole mount of neurula and its vertical section (VS)
• whole mount of tadpole larva of frog
• Transverse section (T .S.) through ear of tadpole
• Transverse section (T .S.) through eyes of tadpole
• Transverse section (T .S.) through the head and gills of tadpole
• Compound microscope
• Lab manual BZYCL-134
• Practical Note book
• Hand lens
• Pen, pencil, erasor and scale.

6.3 STUDY GUIDE


In order to complete your laboratory exercise satisfactorily, you must read in
advance, the unit, entitled “Development of frog” (Unit 14), given in Block 4,
Volume 2 of the theory Core Zoology Course-BZYCT-133 entitled
(Comparative Anatomy and Developmental Biology of Vertebrates which
is part of the B.Sc. General Course (CBCS).

You will study the salient development stages of frog by means of permanent
slides provided by the counsellor and with the help of labelled figures and
diagrams given in the lab manual BZYCL-134.

6.4 DEVELOPMENTAL STAGES OF FROG


Before you start studying the slides, under the compound microscope,
66 carefully read the laboratory exercise thoroughly and study the diagrams of the
Exercise 6 Study of Prepared Sides of the Developmental Stages of Frog
developmental stages provided in it. This will help you to become familiar with
the various developmental processes occurring in frog and to relate them with
the embryological slides provided. First by examining whole mounts of the
embryonic stages under the microscope you will become familiar with the
general and overall changes taking place in frog development. Make well
labelled diagrams of these stages in your practical note book.

In this laboratory exercise you will also be provided with slides of transverse
sections of some developmental stages of frogs. Correlate the description and
figures given in this exercise with study of developmental stages observed by
you in the slides under the microscope. You should be able to correlate the
structures of the whole mount with their respective sections.

Examine carefully the slides provided to you, first under the low power and if
necessary under the high power of the compound microscope and compare
what you have observed with the description and figures provided in the lab
exercise. After viewing one slide you may move to next until you complete
viewing the entire series. In your practical notebook draw, label and write the
description of all the stages of development of frog which you have observed.

Developmental stages to be studied

The developmental stages in the form of whole mounts and sections which
will be examined by you and for which descriptions are provided are listed
below:

1. Unfertilized egg;

2. Fertilized egg;

3. Blastula entire and V.S. of blastula;

4. Gastrula entire and V.S. of late gastrula;

5. Intact neurula and V.S. of neurula;

6. Tadpole larva of frog;

7. T.S. of tadpole through eye region;

8. T.S. of tadpole through auditory vesicles (ears); and

9. T.S. of tadpole through head and gills.

6.4.1 Unfertilized Egg of Frog


i) The female frogs deposits its ova (Fig. 6.1) in water.

ii) The egg is surrounded and covered by three distinct layers which are as
follows:

a) the innermost vitelline membrane which is secreted by the egg


itself;

b) chorion which forms the intermediate layer and is secreted by


follicular cells of the ovary; and
67
BZYCL-134 Laboratory of Comparative Anatomy and Development Biology of Vertebrates

c) the outermost; gelatinous, tertiary egg membrane or albumen


consisting of 3-4 gelatinous rings which is secreted by the wall of
the oviduct.

iii) The eggs are mesolecithal as they contain moderate amounts of yolk.
The frog egg is also telolecithal as the yolk is concentrated in the vegetal
(lower) hemisphere.

iv) The ovum has polarity even before fertilization and so two distinct
regions can be observed: i) The animal hemisphere which contains
pigment melanin and so is the darkly pigmented region, and occupies
more than half of the egg and contains the nucleus; and ii) the vegetal
hemisphere, the light coloured, yolk filled heavier region. The centre of
the animal and vegetal hemispheres are called animal and vegetal pole
respectively.

v) The pigmented region of the egg will form the future animal hemisphere,
while the non- pigmented region will form the future vegetal hemisphere.

vi) The clear cytoplasm and haploid nucleus of the egg are located in the
animal pole. The vegetal hemisphere contains the bulk of the yolk, which
provides the necessary nutrients for embryonic development.

Fig. 6.1: Unfertilized egg of frog.

6.4.2 Zygote: Fertilized Egg


i) The fertilization of the egg by the entry of the sperm into the egg, results
in the formation of zygote.

ii) The centre of the animal and vegetal hemispheres are called the animal
and vegetal pole respectively (Fig. 6.2).

iii) The animal hemisphere contains the diploid nucleus formed by the union
of sperm and egg nucleus.

iv) In the fertilized egg the vitelline membrane is now called fertilization
membrane. Soon after fertilization, the egg secretes a protein material
which absorbs water and swells up, causing the vitelline membrane to lift
68 up from the zygote.
Exercise 6 Study of Prepared Sides of the Developmental Stages of Frog
v) A grey crescent is visible opposite the point of entry of the sperm at the
margin between the animal and vegetal pole, largely in the animal pole.
It is more lightly pigmented than the animal hemisphere. The grey
crescent later forms the posterior and dorsal side of the embryo.

Fig. 6.2: Fertilized egg.

6.4.3 Blastula
(I) Features of whole mount of Blastula

The blastula develops after about the fourth or fifth cleavage and appears in
external appearance as a ball of cells. The cells of the blastula are referred to
as blastomeres (Fig 6.3 a) and are of two different sizes: the smaller,
pigmented blastomeres which are located in the animal hemisphere are the
micromeres, and the larger yolk filled blastomeres are called macromeres and
occupy the vegetal hemisphere. Externally the micromeres of the animal pole
cover most of the blastula.

(II) Features of V.S. of Blastula

i) The blastula internally surrounds a central cavity, the blastocoel which


can only be seen in the section of blastula. A vertical section of the
blastula shows that it contains an excentric blastocoel cavity surrounded
by unequal sized blastomeres (Fig. 6.3 b).

ii) The micromeres at the animal hemisphere form the thin, multilayered
roof over the blastocoel, while the megameres of the vegetal hemisphere
form the floor of the blastocoel.

Fig. 6.3: Blastula: a) whole mount, b) Vertical Section. 69


BZYCL-134 Laboratory of Comparative Anatomy and Development Biology of Vertebrates

6.4.4 Gastrula
(I) Features of whole mount of Gastrula
(i) The blastula is transformed into a gastrula, which appears
externally as a ball of dark pigmented cells, with a circular, light
coloured yolky plug formed of larger vegetal cells (Fig. 6.4a).
(ii) The size of the gastrula is still as small as the zygote (Fig. 6.4).
(II) Features of vertical section of Gastrula
i) Internally the gastrula is seen to be transformed from the single layered
blastula into a three layered structure by the rearrangement of the
cells of the blastula. Gastrulation reorganises the embryo
completely and the gastrula consists of the three germ layers 1)
external ectoderm; 2) The middle mesoderm; and 3) innermost
endoderm. The various organs of the animal are derived from
these three layers. These three layers of the gastrula are well
differentiated and are clearly visible in vertical section (Fig. 6.4b).
ii) The external layer the ectoderm will give rise to the epidermis,
cutaneous glands, nervous system, eye parts and the lining of the
mouth cavity and cloaca.
iii) The middle layer the mesoderm appears as a small area towards
the posterior end and will give rise to musculature, connective
tissue, vascular system, genital organs, excretory organs, skeleton
and notochord.
iv) The inner most layer the endoderm will form the lining of
alimentary canal, liver, pancreas, lung, urinary bladder and
primordial germ cells.
v) Other structures seen in the v.s of blastula are the dorsal lip of
blastopore, yolk plug and ventral lip of the blastopore.
vi) The notochord or chorda cells and neural plates are well
differentiated and lie at the dorsal side.
vii) Blastocoel is reduced due to the newly developed archenteron
cavity which lies towards the dorsolateral side of the anterior part
of the embryo. The archeneteron will develop into gut in the future.

Fig. 6.4: Gastrula: a) whole mount; b) Vertical Section.


70
Exercise 6 Study of Prepared Sides of the Developmental Stages of Frog
6.4.5 Neurula
(I) Features of Whole mount of Neurala
i) The embryo is called neurula (Fig. 6.5) when the two neural plates of
the gastrula join to form a neural tube (which will form the nervous
system of the frog) along the longitudinal axis.
ii) Externally the neural tube in the embryo appears as a thickened ridge
along the longitudinal axis.
(II) Features of V.S. of Neurala
i) In vertical section of the neurula, the neural tube which is of ectodermal
origin lies above the notochord.
ii) The notochord, a rod-like structure arises from the mesoderm in the
middorsal region and lies below the neural tube and above the
archenteron cavity. The notochord runs along the longitudinal axis
of the embryo.
iii) On either side of the neural tube and notochord extensive mesodermal
tissues are present.
iv) The mesodermal tissues which flank the neural tube form the somite
mesoderm.
v) The lateral mesoderm which is a continuation of the somite mesoderm
appears split into 1) The splanchnic mesoderm which lies nearest
to the gut endoderm; and 2) the somatic mesoderm which lies
nearest to the endoderm.
vi) A new fluid filled cavity the coelom is present between the splanchnic
and somatic mesoderm.

Fig. 6.5: Neurula: (a) whole mount (b) Vertical Section.

6.4.6 Features of Tadpole of a Frog: Whole Mount


i) The eggs of the frog hatch in the water and release free living
herbivorous larva (Fig. 6.6).
ii) The larva measures about 5-7 mm in length.
iii) The body of the larva is differentiated into a head, a body proper and a
tail with fin. 71
BZYCL-134 Laboratory of Comparative Anatomy and Development Biology of Vertebrates
iv) The larva contains a head with mouth, sucker, external gills, rudiments
of eye and olfactory pits which are visible externally. The larva also
contains the gut, anus and myotomes (muscles) which are present
internally.
v) Mouth of larva bears horny jaws or teeth.
vi) Three pairs of external feathery gills can be seen. These act as the
functional respiratory organs.
vii) Tail is long and bears the tail fin on its ventral and dorsal surface.
viii) The herbivorous larva feeds on vegetation and has a coiled intestine.
ix) The larva metamorphoses into an adult amphibians (which live both on
land and water) frog.

Fig. 6.6: Whole mount of frog tadpole.

6.4.7 Features of Transverse Section of Tadpole


through the Eye Region
i) The paired eyes in the frog tadpole develop very early and are of
ectodermal origin. They are the most conspicuous and protuberant
structures of the tadpole.
ii) Eyes develop from the optic vesicles which originate from a pair of
diverticula given off from the prosencephalon of the brain on each side.
iii) A transverse section (T.S.) of the tadpole through the eye region
(Fig. 6.7) shows the forebrain in the centre on the dorsal surface.
iv) The paired eyes are seen on the lateral sides of the brain and are
composed of lens, sensory layer of retina and eye muscles.
v) The single pharynx lies in the centre of the transverse section.
vi) Hyoid arch lies just below the pharynx in the transverse section.
vii) The paired palatoquardate bars are placed dorsal to the pharynx on
each lateral side.
viii) Meckel's cartilage is present on each lateral side of the pharynx.
ix) Other structures seen in the section are jaw muscles, trabaculae,
72 epidermis and connective tissue.
Exercise 6 Study of Prepared Sides of the Developmental Stages of Frog

Fig. 6.7: Transverse section of tadpole of a frog through the eye region.

6.6.8 Features of Transverse Section of Tadpole


through the Auditory Vesicles
The transverse section of tadpole through the ear region shows the following
structures:

i) The paired ears (auditory vesicles) develop as a pair of auditory pits


from the epiblast on the lateral sides of the hind brain.

ii) The auditory vesicles develop at an early tadpole stage and are found on
the dorso lateral side of the section, below the hind brain (Fig. 6.8).

iii) The auditory pits become vesicle-like and enclose the middle ear. The
semicircular canals develop as outgrowths from the wall of the auditory
vesicles.

iv) The single median, medulla oblongata of the hind brain encloses the
fourth ventricle and is located in the middorsal region of the section
between the two auditory capsules.

v) The notochord lies below the medulla oblongata.

vi) The branchial chamber containing the laryngeal chamber lies below the
notochord and auditory vesicles.

vii) The pericardial cavity enclosing the developing auricle (atrium) and
ventricle of the heart is located on the ventral side of the section, below
the laryngeal chamber.

Fig. 6.8: Transverse section of tadpole of a frog through the Audltory Vesicles
(ears). 73
BZYCL-134 Laboratory of Comparative Anatomy and Development Biology of Vertebrates

6.4.9 Features of Transverse Section of Tadpole through


Head and Gill Region
Transverse section of the tadpole (Fig. 6.9) through the head and gill region
shows the following details:

1. The median medulla oblongata enclosing the fourth ventricle lies in the
middorsal position.

2. The paired membranous labyrinth are present on both the lateral sides
of the medulla oblongata.

3. The notochord lies just below the medulla oblongata.

4. The pharynx lies in the centre of the section.

5. Larynx is seen in continuation with pharynx.

6. The heart enclosed in the pericardium is seen just below the larynx.

7. The branchial arches and internal gills are present on the lateral sides of
the pericardium.

8. The lateral dorsal aorta is located above the pharynx.

9. Lymphatic spaces lie on both the lateral side

Fig. 6.9: Transverse section of frog tadpole through Head and Gills.

6.5 TERMINAL QUESTIONS


1. Explain the necessity for the three jelly coats in the frog ovum.

2. With the help of labeled diagrams show the differences between the
unfertilized and fertilized frog eggs.

3. From which germ layer does the nervous system develop?


74

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